This invention relates to a system for transporting bioactive molecules into eukaryotic cells. More particularly, the invention relates to a composition and a method for delivering negatively charged bioactive molecules, i.e. nucleic acids, into a host cell using a biodegradable, mixed polymeric micelle comprising an amphiphilic polyester-polycation copolymer and an amphiphilic polyester-sugar copolymer.
Early efforts to identify methods of delivering nucleic acids into tissue culture cells began in the mid-1950""s. H. E. Alexander et al., 5 Virology 172-173 (1958). Since then, steady progress has been made toward improving delivery of functional DNA, RNA, and antisense oligonucleotides (RNA function inhibitors) in vitro and in vivo. Substantial progress has been achieved during the last two decades due to the convergence of transfection technology and recombinant DNA technology in the late 1970""s. This convergence began when calcium phosphate and diethylaminoethyldextran were applied to the expression of recombinant plasmids in cultured mammalian cells. P. J. Southern et al., 1 J. Mol. Appl. Gen. 327-341 (1982). Presently, delivery and expression of nucleic acids has become a topic that continues to capture scientific attention.
Some success has been achieved in delivering functional, non-replicating plasmids in vitro, however, the current methods for delivering functional, non-replicating plasmids in vivo are in their infancy. Transfection techniques include methods using insoluble inorganic salts, F. Graham, 52 Virology 456-462 (1973), cationic lipids, E. R. Lee et al., 7 Human Gene Therapy 1701-1717 (1996), cationic polymers, B. A. Demeneix et al., 7 Human Gene Therapy 1947-1954 (1996); A. V. Kabanov et al., 6 Bioconjugate Chem. 7-20 (1995); E. Wagner, 88 Proc. Natl. Acad. Sci. USA 4255-4259 (1991), viral vectors, A. H. Jobe, 7 Human Gene Therapy 697-704 (1996); J. Gauldie, 6 Current Opinion in Biotechnology 590-595 (1995), cell electroporation, U.S. Pat. No. 5,501,662 (1996); U.S. Pat. No. 5,273,525 (1993), and microinjection, U.S. Pat. No. 5,114,854 (1992). Each of the above-listed methods has specific disadvantages and limitations. The most widely studied gene transfer carriers are viral vectors, including retrovirus, adenovirus, adeno-associated virus, and herpes. virus systems. Viral vectors have shown a high transfection efficiency compared to non-viral vectors, but their use in vivo is severely limited. Their drawbacks include targeting only dividing cells, random insertion into the host genome, risk of replication, and possible host immune reaction. J. M. Wilson, 96 J. Clin. Invest. 2547-2554 (1995).
Compared to viral vectors, nonviral vectors are easy to manufacture, less likely to produce immune reactions, and will not produce replication reactions. Dimethylaminoethyldextran-, calcium phosphate- and polycation-mediated transfection procedures have been used for tissue culture cells in the laboratory. Under some conditions, transfection efficiencies of close to 100% of the cells have been obtained in vitro. In general, however, nonviral vectors have been found to be ineffective for in vivo introduction of genetic material into cells and have resulted in relatively low gene expression. Various cationic amphiphiles have been extensively investigated and added to liposome formulations for the purpose of gene transfection. F. D. Ledley, 6 Human Gene Therapy 1129-1144 (1995). To date, a cationic lipid system has been regarded as the most promising DNA transfection protocol, because it overcomes the problems with using viral vectors. However, transfection efficiency using cationic lipids is still not as high as with viral vectors, and complaints about cytotoxicity have been raised. Therefore, continuing investigation is required to find a new gene transfer protocol.
In view of the foregoing, it will be appreciated that development of a bioactive molecule delivery system, especially a gene delivery system for in vivo use that is both safe and efficient would be a significant advancement in the art.
It is an object of the present invention to provide a composition and method for delivering bioactive molecules, i.e. nucleic acids, into cells.
It is also an object of the invention to improve delivery efficiency by providing a particulate carrier wherein particle size and charge density are easily controlled by multivariate means.
It is another object of the invention to provide a composition and a method for bioactive molecule delivery wherein the carrier is biodegradable and biocompatible.
These and other objects are addressed by providing a carrier for delivery of a selected bioactive molecule into a host cell, the carrier comprising a mixture of an amphiphilic polyester-polycation copolymer and an amphiphilic polyester-sugar copolymer. In a preferred embodiment of the invention, the polyester-polycation copolymer comprises about 5 to 95% by weight of the carrier. The polyester-sugar copolymer also preferably comprises about 5 to 95% by weight of the carrier. The polyester polyeation copolymer can be either a diblock copolymer comprising a hydrophobic polyester block bonded to a hydrophilic polycation block by an amide linkage or a graft copolymer comprising a hydrophobic polyester portion and a hydrophilic cation portion. The polyester is preferably a member selected from the group consisting of poly(L-lactic acid), poly(D-lactic acid), poly(D-,L-lactic acid), poly(glycolic acid), poly(L-lactic-co-glycolic acid), poly(D-lactic-co-glycolic acid), poly(xcex5-caprolactone), polybutyrolactone, and polypropiolactone. More preferably, the polyester is poly(L-lactic acid). In a preferred embodiment of the invention the polyester has a molecular weight of about 500 to 10,000. The polycation is preferably a member selected from the group consisting of poly(L-serine ester), poly(D-serine ester), poly(L-lysine), poly(D-lysine), polyomithine, and polyarginine, and more preferably is poly(L-serine ester). In a preferred embodiment of the invention the polycation has a molecular weight of about 500 to 10,000.
The polyester-sugar copolymer comprises a hydrophobic polyester segment and a hydrophilic sugar segment. The polyester segment is preferably a member selected from the group consisting of poly(L-lactic acid), poly(D-lactic acid), poly(D-,L-lactic acid), poly(glycolic acid), poly(L-lactic-co-glycolic acid), poly(D-lactic-co-glycolic acid), poly(xcex5-caprolactone), polybutyrolactone, and polypropiolactone, and more preferably is poly(L-lactic acid). In a preferred embodiment of the invention the polyester segment has a molecular weight of about 500 to 10,000. The sugar segment can comprise either a polysaccharide or a glycosylated polymer. Such a glycosylated polymer preferably comprises at least one sugar moiety selected from the group consisting of galactose, glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose, trehalose, maltose, galactosamine, glucosamine, galacturonic acid, glucuronic acid, gluconic acid, and lactobionic acid, and more preferably is lactobionic acid. The polymer moiety of the glycosylated polymer is preferably selected from the group consisting of poly(L-serine ester), poly(D-serine ester), poly(L-lysine), poly(D-lysine), polyornithine, and polyarginine and more preferably is poly(L-serine ester), and more preferably is poly(L-lysine).
The carrier can optionally further comprise a copolymer comprising (a) a hydrophobic portion, (b) a hydrophilic portion bonded to the hydrophobic portion, and (c) a functional moiety coupled to the hydrophilic portion, wherein the functional moiety is a member selected from the group consisting of ligands, fusogenic agents, lysosomotrophic agents, nucleus localizing signals, and mixtures thereof. Such a ligand is preferably selected from the group consisting of transferrin, epidermal growth factor, insulin, asialoorosomucoid, mannose-6-phosphate, mannose, LewisX, sialyl LewisX, N-acetyllactosamine, galactose, glucose, and thrombomodulin; the fusogenic agent is preferably selected from the group consisting of polymixin B and hemaglutinin H2; and the nucleus localization signal is preferably T-antigen.
The carrier of the present invention is particularly useful for delivery of a negatively charged molecule into a host cell, such as negatively charged drugs, or nucleic acids. One preferred embodiment of the present invention is a composition for gene delivery.
A composition for delivery of a selected bioactive molecule into a host cell comprises a mixed-polymeric-micelle/nucleic-acid complex in an aqueous medium, wherein the mixed-polymeric-micelle/bioactive molecule complex comprises (a) a mixture of an amphiphilic polyester-polycation copolymer and an amphiphilic polyester-sugar copolymer, and (b) an effective amount of the selected nucleic acid.
A method for delivering a selected bioactive molecule into a host cell comprises administering an effective amount of a mixed-polymeric-micelle/bioactive molecule complex in an aqueous medium, wherein the mixed-polymeric-micelle/bioactive molecule complex comprises (a) a mixture of an amphiphilic polyester-polycation copolymer and an amphiphilic polyester-sugar copolymer comprising a sugar moiety, and (b) an effective amount of said selected bioactive molecule, such that the complex contacts the host cell and the sugar moiety triggers receptor mediated endocytosis of the mixed-polymeric-micelle/bioactive molecule complex, thus delivering the bioactive molecule into the host cell.